The combination of combinatorial chemistry, sequencing of the genomes of many species and relationships between genotype and physical and biological traits has greatly expanded the need to perform determinations of different events. The multiplicity of new compounds that can be prepared using various forms of combinatorial chemistry and the numerous targets involving wild-type and mutated genes, had extraordinarily increased the number of determinations of interest in developing compounds having biological activity. These compounds include drugs, biocides, pesticide resistance, disease organism resistance and the like. In addition, the interest in discriminating between different genomes, relating specific mutations to phenotypes, defining susceptibilities to various environmental effects in relation to single nucleotide polymorphisms, and identifying the genomes of organisms to provide better defenses against the organisms has expanded the need for rapid, inexpensive devices and methodologies for carrying out these and other determinations.
Recently, microfluidic arrays have been developed which allow for a multiplicity of reservoirs and channels to be associated with a small card or chip, where by using high voltages, various operations can be performed. The arrays provide for individual networks, which exist in combination on a single chip, so that a plurality of determinations may be performed concurrently and/or consecutively. By having channels that have cross-sections in the range of about 500 to 5000 μm2, operations can be carried out with very small volumes. In addition, by having very sensitive detection systems, very low concentrations of a detectable label may be employed. This allows for the use of very small samples and small amounts of reagents, which have become increasingly more sophisticated and expensive. Microfluidic arrays offer the promise of more rapid throughput, increasingly smaller times to a determination and increasingly smaller amounts of sample and reagents being required.
The use of microfluidic arrays, however, has its challenges. The microfluidic arrays are desirably made in molded plastic, so as to provide a reduced cost of the chip. By molding the chip and providing for ridges on a mold to form the channels, the channels may not run true and may be displaced from their proper positions, as well as being slightly curved rather than perfectly straight. In addition, the plastic frequently autofluoresces. Since the frequently used label is a fluorescent label, the signal from the label must be able to be distinguished from the autofluorescent signal. There is the problem of how to obtain a reliable fluorescent signal, in effect compromising maximizing the signal from the detectable label while minimizing the background signal.
In addition, the channel walls are not orthogonal to the cover plate, so that the depth of the irradiation may vary, depending upon the site of entry of the excitation beam into the channel. Where the excitation beam encounters the wall, the signal is degraded due to the reduced number of fluorophores which are excited and the excitation of the fluorophores in the wall. Therefore, precise positioning of the excitation beam in the channel is necessary for reproducible and accurate results.